mGluR5 Positive Allosteric Modulation Prevents MK-801 Induced Increases in Extracellular Glutamate in the Rat Medial Prefrontal Cortex.

Potentiation of metabotropic glutamate receptor subtype 5 (mGluR5) function produces antipsychotic-like and pro-cognitive effects in animal models of schizophrenia and can reverse cognitive deficits induced by N-methyl-D-aspartate type glutamate receptor (NMDAR) antagonists. However, it is currently unknown if mGluR5 positive allosteric modulators (PAMs) can modulate NMDAR antagonist-induced alterations in extracellular glutamate levels in regions underlying these cognitive and behavioral effects, such as the medial prefrontal cortex. We therefore assessed the ability of the mGluR5 PAM, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), to reduce elevated extracellular glutamate levels induced by the NMDAR antagonist, dizocilpine (MK-801), in the medial prefrontal cortex. Male Sprague-Dawley rats were implanted with a guide cannula aimed at the medial prefrontal cortex and treated for ten consecutive days with MK-801 and CDPPB or their corresponding vehicles. CDPPB or vehicle was administered thirty minutes before MK-801 or vehicle each day. On the final day of treatment, in vivo microdialysis was performed, and samples were collected every thirty minutes to analyze extracellular glutamate levels. Compared to animals receiving only vehicle, administration of MK-801 alone significantly increased extracellular levels of glutamate in the mPFC. This effect was not observed in animals administered CDPPB before MK-801, nor in those administered CDPPB alone, indicating that CDPPB decreased extracellular glutamate release stimulated by MK-801. Results indicate that CDPPB attenuates MK-801 induced elevations in extracellular glutamate in the medial prefrontal cortex. This effect of CDPPB may underlie neurochemical adaptations associated with the pro-cognitive effects of mGluR5 PAMs in rodent models of schizophrenia.

Direct administration of ifenprodil and citalopram into the nucleus accumbens inhibits cue-induced nicotine seeking and associated glutamatergic plasticity.

Nicotine use disorder has been associated with glutamatergic alterations within the basal ganglia that might contribute to relapse. Specifically, initiation of cue-induced nicotine seeking produces rapid, transient synaptic potentiation (t-SP) in nucleus accumbens core (NAcore) medium spiny neurons (MSNs), defined as increases in spine head diameter and AMPA to NMDA current ratios (A/N). Ifenprodil, which inhibits nicotine reinstatement when administered systemically, antagonizes GluN2B-containing NMDA receptors, has affinity for serotonin receptors, and blocks serotonin transporters (SERT). The mechanisms underlying its therapeutic efficacy, however, remain unknown. Using pharmacological and genetic approaches, the current study examined the role of NAcore GluN2B receptors as well as SERT in mediating cue-induced nicotine seeking and associated MSN structure and physiology. Prior to reinstatement, rats received intra-NAcore injections of either ifenprodil, citalopram or artificial cerebral spinal fluid (15 min prior), or GluN2B or control siRNAs (3 consecutive days prior). Rats were sacrificed after a 15-min cue-induced reinstatement session for dendritic spine analysis, western blotting or whole-cell electrophysiology. Intra-NAcore ifenprodil blocked nicotine-seeking behavior and promoted a higher frequency of shorter spines on MSN dendrites. However, a decrease in membrane-bound GluN2B receptor expression did not prevent cue-induced nicotine seeking or associated MSN cell physiology. Interestingly, intra-NAcore citalopram, an SSRI, prevented cue-induced nicotine seeking. Together, these results indicate that the therapeutic effects of ifenprodil on cue-induced nicotine seeking may, in part, be due to its actions at SERT rather than GluN2B, which may be specific to nicotine-seeking as opposed to other drugs of abuse.

AMPA receptor and metabotropic glutamate receptor 1 adaptations in the nucleus accumbens core during incubation of methamphetamine craving.

Cue-induced drug craving progressively intensifies after withdrawal from self-administration of cocaine, methamphetamine, and other drugs of abuse, a phenomenon termed incubation of craving. For cocaine and methamphetamine, expression of incubated craving ultimately depends on strengthening of nucleus accumbens (NAc) synapses through an accumulation of high conductance Ca2+-permeable AMPA receptors (CP-AMPARs) that is detectable with electrophysiological approaches. This study sought to further characterize glutamate receptor adaptations in NAc core during methamphetamine incubation. Previous biochemical studies revealed that the CP-AMPARs accumulating after cocaine incubation are mainly homomeric GluA1 receptors and that their accumulation is reflected by increased cell surface GluA1. Here, for methamphetamine, we observed no significant change in surface or total GluA1 (GluA2 and GluA3 were also unchanged). Nonetheless, GluA1 translation was elevated after incubation of methamphetamine craving, as recently found for cocaine. Additionally, for cocaine, we previously observed a withdrawal-dependent decrease in mGlu1 surface expression that precedes and enables CP-AMPAR accumulation and incubation of craving, reflecting weakening of mGlu1-dependent mechanisms that normally limit synaptic CP-AMPAR levels in the NAc core. Here, we observed no change in surface or total mGlu1 protein or its coupling to Homer scaffolding proteins after methamphetamine withdrawal, nor did elevation of mGlu1 tone through repeated injections of an mGlu1-positive allosteric modulator delay incubation of craving. These findings suggest a common role for increased GluA1 translation, but not decreased mGlu1 function, in the incubation of methamphetamine and cocaine craving. We speculate that increased GluA1 translation near synapses may drive formation and synaptic insertion of homomeric GluA1 receptors in the absence of detectable changes in GluA1 protein levels.

α6 subunit-containing nicotinic receptors mediate low-dose ethanol effects on ventral tegmental area neurons and ethanol reward.

Dopamine (DA) neuron excitability is regulated by inhibitory GABAergic synaptic transmission and modulated by nicotinic acetylcholine receptors (nAChRs). The aim of this study was to evaluate the role of α6 subunit-containing nAChRs (α6*-nAChRs) in acute ethanol effects on ventral tegmental area (VTA) GABA and DA neurons. α6*-nAChRs were visualized on GABA terminals on VTA GABA neurons, and α6*-nAChR transcripts were expressed in most DA neurons, but only a minority of VTA GABA neurons from GAD67 GFP mice. Low concentrations of ethanol (1-10 mM) enhanced GABAA receptor (GABAA R)-mediated spontaneous and evoked inhibition with blockade by selective α6*-nAChR antagonist α-conotoxins (α-Ctxs) and lowered sensitivity in α6 knock-out (KO) mice. Ethanol suppression of VTA GABA neuron firing rate in wild-type mice in vivo was significantly reduced in α6 KO mice. Ethanol (5-100 mM) had no effect on optically evoked GABAA R-mediated inhibition of DA neurons, and ethanol enhancement of VTA DA neuron firing rate at high concentrations was not affected by α-Ctxs. Ethanol conditioned place preference was reduced in α6 KO mice compared with wild-type controls. Taken together, these studies indicate that relatively low concentrations of ethanol act through α6*-nAChRs on GABA terminals to enhance GABA release onto VTA GABA neurons, in turn to reduce GABA neuron firing, which may lead to VTA DA neuron disinhibition, suggesting a possible mechanism of action of alcohol and nicotine co-abuse.

Diolistic Labeling and Analysis of Dendritic Spines.

Dendritic spines are diverse and plastic components of the neuronal cell apparatus and are highly responsive to trophic factors during both development and adulthood. Diolistic labeling of neurons with lipophilic fluorescent dyes, coupled with advanced high-resolution microscopy methods, provides robust labeling of dendritic spines for assessment of their density and morphology. Here, we describe a method for labeling of dendritic spines using diolistic labeling in ex vivo brain slices, visualization using confocal laser scanning microscopy, deconvolution, and analysis using the Surpass module of Bitplane Imaris software.

Estradiol Facilitation of Cocaine Self-Administration in Female Rats Requires Activation of mGluR5.

In comparison to men, women initiate drug use at earlier ages and progress from initial use to addiction more rapidly. This heightened intake and vulnerability to drugs of abuse is regulated in part by estradiol, although the signaling mechanisms by which this occurs are not well understood. Recent findings indicate that within the nucleus accumbens core, estradiol induces structural plasticity via membrane-localized estrogen receptor α, functionally coupled to metabotropic glutamate receptor subtype 5 (mGluR5). Hence, we sought to determine whether mGluR5 activation was essential for estradiol-mediated enhancement of cocaine self-administration. Ovariectomized (OVX) female rats were allowed to freely self-administer cocaine under extended access conditions (6 h/d) for 10 consecutive days. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) or vehicle was administered before estradiol (or oil), on a 2 d on/2 d off schedule throughout the extended access period. MPEP treatment prevented the estradiol-dependent enhancement of cocaine self-administration in OVX females. In a separate experiment, potentiation of mGluR5 function with the positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (in the absence of estradiol treatment) failed to increase cocaine self-administration. These data suggest that mGluR5 activation is necessary for estradiol-mediated enhancement of responses to cocaine, but that direct mGluR5 activation is insufficient to mimic the female response to estradiol. Building on previous studies in male animals, these findings further highlight the therapeutic potential of mGluR5 antagonism in the treatment of addiction and suggest that there may be added therapeutic benefit in females.

Chronic stress disrupts fear extinction and enhances amygdala and hippocampal Fos expression in an animal model of post-traumatic stress disorder.

Chronic stress may impose a vulnerability to develop maladaptive fear-related behaviors after a traumatic event. Whereas previous work found that chronic stress impairs the acquisition and recall of extinguished fear, it is unknown how chronic stress impacts nonassociative fear, such as in the absence of the conditioned stimulus (CS) or in a novel context. Male rats were subjected to chronic stress (STR; wire mesh restraint 6 h/d/21d) or undisturbed (CON), then tested on fear acquisition (3 tone-footshock pairings), and two extinction sessions (15 tones/session) within the same context. Then each group was tested (6 tones) in the same context (SAME) or a novel context (NOVEL), and brains were processed for functional activation using Fos immunohistochemistry. Compared to CON, STR showed facilitated fear acquisition, resistance to CS extinction on the first extinction day, and robust recovery of fear responses on the second extinction day. STR also showed robust freezing to the context alone during the first extinction day compared to CON. When tested in the same or a novel context, STR exhibited higher freezing to context than did CON, suggesting that STR-induced fear was independent of context. In support of this, STR showed increased Fos-like expression in the basolateral amygdala and CA1 region of the hippocampus in both the SAME and NOVEL contexts. Increased Fos-like expression was also observed in the central amygdala in STR-NOVEL vs. CON-NOVEL. These data demonstrate that chronic stress enhances fear learning and impairs extinction, and affects nonassociative processes as demonstrated by enhanced fear in a novel context.

Hypothesizing that designer drugs containing cathinones ("bath salts") have profound neuro-inflammatory effects and dangerous neurotoxic response following human consumption.

Consensus in the most recent literature indicates that psychoactive "bath salts" is a relatively new drug-combination that was added to Schedule I classification in October 2011. Common ingredients include the cathinone analogs: mephedrone and methylenedioxypyrovalerone (MDPV). The mechanism of action of these synthetic cathinone analogs has not yet been well studied. We propose an intensive systematic investigation to determine the potential for cathinones to produce neurotoxic effects in various brain regions. In spite of a lack of evidence, for neurotoxicity there are number of horrific cases now on record that suggest intensification of research is needed. For example, a suicide by hanging had high 3,4-MDPV concentration while a driver under the influence had the highest reported methylone (MEPH) concentration. More interestingly, there have been consistent case reports indicating delayed responses, including: severe agitation with possible psychosis, suicidal ideation, rhabdomyolysis, hypertension, tachycardia, and death. In animal studies, amphetamine (AMPH), methamphetamine (METH) and cocaine release dopamine (DA), similarly to the action of cathinone and particular cathinone analogues. Two components of bath salts, MEPH and MDPV produce opposite effects at human dopamine transporter (hDAT) comparable to METH and cocaine, respectively. Moreover, it has already been found by others that MEPH is almost as potent as METH; and MDPV is much more potent than cocaine with longer lasting effects. It has been conjectured correctly that bath salts containing MDPV and MEPH (or a similar drug) might be expected both, to initially release DA and subsequently prevent its reuptake via hDAT. The null hypothesis, that cathinones do not cause neurotoxicity to dopamine nerve endings of the striatum, seems parsimonious and requires intensive investigation. Our hypothesis is that when consumed by humans, cathinones may induce neurotoxic pathways involving the neuro-glial-microglia and/or specific inflammation, that may help explain the clinically observed delayed response. We intend to explore this hypothesis utilizing a novel proteomic and biomarker technique developed by scientists at the McKnight Brain Institute, University of Florida as well as magnetic-resonance imaging across pre-frontal orbital cortex-cingulate gyrus and mesolimbic pathways of the brain of rodents.